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  • C12N2310/50—Physical structure
  • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

• the present invention relates to alteration of gene expression with a stable DNA construct, conveniently referred to as a cassette, into which a nucleic acid sequence is incorporated for use as a template for subsequent production of that sequence in a prokaryotic or eukaryotic host cell, and a system for expression of that sequence within eukaryotic host cells without (or with minimal) flanking sequences
• the construct, or cassette includes inverted tandem repeats that form the stem of a stem-loop intermediate that functions m vivo to cause expression of the sequence, referred to as the sequence of interest, as a single stranded DNA (ssDNA) sequence that binds to or otherwise interacts with a target gene to alter expression of that gene
• the expression system of the present invention removes most or all contiguous plasmid (or other vector) sequences from the ssDNA either by stem-loop formation with subsequent termination of a reverse transc ⁇ ption reaction by the stem or by cleavage of the stem-loop intermediate
• Antisense gene therapy has been successfully used in a variety of applications to down regulate gene function Jain, K K , Handbook of Gene Therapy, New York Hofgrefe & Huber Publishing (1998) To date, however, such therapy has been characterized by a number of disadvantages and limitations, some serious, that have decreased the utility of this type of therapy, including the short half-life of the antisense molecule, non-specific effects, uncertainties as to the mode of action of the antisense sequence, and potential toxic effects in animal studies
• ODNs antisense o gonucleotides
• ODNs antisense o gonucleotides
• their analogs must be administered intravenously, which involves problems in cell uptake and distribution (Cossum, P A , et al , Disposition of the C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats, 267 J Pharmacol Exp Ther 1 181-1 190 (1993), Sands, H , et al
• Oligodeoxynucleotides Antisense inhibitors of gene expression, in J S Cohen (Ed ), Boca Raton CRC Press, p 79 (1989)) and are eliminated rapidly (Chen, T L , et al , Disposition and metabolism of oligodeoxynucleoside methylphosphonate following a single i v injection in mice , 18 Drug Metab Dispos. 815-818 (1990))
• Ribozymes comprise only RNA molecules, which can catalyze the cleavage of specific mRNA sequences, and are thought to be potentially more efficient than antisense ODNs because of their catalytic capability (Woolf, T M , To cleave or not to cleave Ribozymes and antisense, 5 Antisense Res Dev 227-232 ( 1995)) Ribozymes have been used as inhibitors of gene expression and viral replication (Jain, supra (1998)) Unlike antisense ODNs, ribozymes can be delivered either endogenously, such as by using viral vectors, or exogenously However, ribozymes have limited stability due to degradation by RNases in vivo (Jain, supra ( 1998))
• an object of the present invention to provide a DNA construct that directs the synthesis of ssDNA containing a sequence that specifically cleaves mRNA targets in vivo to alter the expression of the gene producing that target mRNA
• ssDNA including such a DNA enzyme sequence of any desired nucleotide sequence within eukaryotic cells without undesirable intervening or flanking nucleotide bases so as to preserve the enzymatic function of the ssDN A against a target nucleic acid, for use in altering expression of a gene including that target nucleic acid.
• Another object of the present invention is to provide a method, and a DNA construct utilized in such methods, for production of ssDNA within eukaryotic cells that contains DNA enzyme sequences for overcoming the significant problems encountered by the use of standard oligonucleotide delivery methods for therapeutic purposes
• Another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA of any nucleotide sequence /// vivo that functions as (but is not limited to) an inhibitory nucleic acid for, for instance, binding to mRNA in an anti-sense fashion to down regulate a gene product or a viral gene product of interest or binding to and inhibiting a specific cellular function, for instance, by binding to proteins that recognize a nucleic acid sequence
• Another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA designed to favor binding to duplex (native DNA) to form triplex structures that may interfere with normal gene transcription and regulation.
• Another object of the present invention is to produce ssDNA within eukaryotic cells for the purpose of disrupting one or more cell functions
• Yet another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA into which secondary structures are designed so that the ssDNA oligonucleotides bind to and/or otherwise inhibit or activate various cellular functions that rely on the catalytic action of a protein or on nucleic acid protein interaction such as transcription, translation, and DNA replication
• Another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA in vivo for site-directed mutagenesis or gene knockout for therapeutic applications
• Another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA of precisely defined nucleotide composition that favors site-specific insertion into a genome for therapeutic purposes
• Yet another object of the present invention is to provide a method, and a DNA construct, for producing ssDNA that is complimentary to any endogenous nucleic acid target for use in altering expression of a gene including the nucleic acid sequence target
• Another object of the present invention is to provide a method, and DNA expression utilized in such a method, for m vivo production of ssDNA including a sequence exhibiting catalytic activity against mRNA targets for transfection into eukaryotic cells that overcomes the obstacles to delivery of direct administration of ssDNA by lipofection, direct cellular uptake, and/or microinjection
• Another object of the present invention is to provide all enzymatic functions that are necessary to produce ssDNA in vivo containing a sequence with enzymatic activity against a target mRNA of choice within a single or dual plasmid expression system
• Another object of the present invention is to provide a method, and pharmacologically acceptable compositions, for delivering an inhibitory nucleic acid sequence including a sequence with enzymatic activity to target cells in a manner which produces a therapeutic effect
• This listing of the objects of the present invention is not intended to be a list of all the objects of this invention
• There are a vast number of other cellular functions that are mediated by the cellular genome which, in the interest of brevity and practicality, are not mentioned here and which are amenable to regulation by m vivo production of ssDNA
• exonucleases digest ssDNA much more aggressively than double-stranded DNA (dsDNA) Consequently, another object of the present invention is to provide a ssDNA construct, and a method of producing that construct in vivo, that is not as susceptible to degradation by native exonucleases in the cell as dsDNA It can be seen from this illustration that this list of some of the objects of the present invention is provided for exemplification and
• a method of altering expression of an endogenous nucleic acid target sequence in a target cell comprising the steps of introducing a cassette comprised of a sequence of interest flanked by an inverted tandem repeat and a 3' primer binding site (PBS) into a target cell and reverse transcribing the mRNA transcript of the cassette from the PBS to release a single- stranded cDNA transcript in the cell
• the sequence of interest is comprised of a nucleic acid sequence that produces a sequence of nucleic acids that binds to an endogenous target nucleic acid sequence when reverse transcribed to alter expression of the target sequence
• Figure 1 is a schematic illustration of a production of ss-cDNA in a host cell in accordance with the present invention
• Figure 2 is a schematic illustration of the stem-loop intermediate formed by the method illustrated in Fig 1
• FIG. 3 is a schematic illustration of the pssXA plasmid comprising a first component of a first embodiment of the expression system of the present invention
• RT reverse transcriptase
• Mboll genes were subcloned into the mammalian expression vector pBK-RSV (Stratagene) and expressed as a single polypeptide.
• the RT and Mboll domains are separated by a histidine-rich linker
• Figures 4A and 4B are schematic illustrations of the pssXB plasmid comprising a second component of the first embodiment of the expression system of the present invention (Fig.
• Figure 5 A is a schematic illustration of the pssXC plasmid comprising a second embodiment of the expression system of the present invention and including the 10-23 D ⁇ A enzyme sequence illustrated schematically in Fig. 5B
• Figures 6A and 6B represent schematic illustrations of the pssXD plasmid comprising a third embodiment of the expression system of the present invention Fig. 6 A) and an elarged portion of the pssXD plasmid (Fig 6B)
• Figure 7 shows the result of a PCR assay for RT activity in a pssXA transfected cell lysate Lanes 1 and 2 A549 cells transiently transfected with the pBK-RSV vector, lanes 3 and 4 A549 cells transiently infected with pssXA, lanes 5 and 6 A549 cells stably transfected with pssXA (E10) Before PCR amplification, reverse transcription reaction was carried out for 10 (lane 1 , 3, and 5) or 30 minutes (lane 2, 4, and 6), repectively, at 37°C
• Figure 8 shows the result of an assay for detection of ssD ⁇ A by PCR analysis
• E10 cells transfected with pssXB-I 2 E10 cells transfected with pssXB-II, 3 E10 cells; 4 A549 cells
• Figure 10 shows a bar graph quantitating a Northern blot of a ssDNA- producing vector constructed in accordance with the present invention producing an antisense sequence against c-raf kinase.
• Figure 1 1 shows the results of a dot blot analysis for detection of ssDNA in A549 cells transfected with control pssXD-I or pssXD-II containing the c-raf DNA enzyme sequence No detectable signal was produced in the presence of S 1 nuclease due to the specific degradation of ssDNA enzyme by S 1 nuclease
• Figure 12 shows the results of quantitative RT-PCR to determine whether ssDNA expressed in A549 cells transfected with pssXD-II altered c-raf mRNA levels
• Figure 13 shows the results of a Western blot for suppression of c-raf protein expression in A549 cells transfected with pssXD-I or pssXD-II Lane 1 pssXD-II, Lane 2 control pssXD-I, Lane 3 untransfected cells
• Figure 14 shows the results of a Western blot for genomic DNA cleavage for induction of cell apoptosis by suppression of c- af gene expression Lane 1 pssXD- II, Lane 2 control pssXD-I, Lane 3 untransfected cells
• Figure 15 shows the results of a Western blot for P ARP cleavage for induction of cell apoptosis by suppression of c-raf gene expression
• Lane 1 pssXD-II Lane 2 control pssXD-I
• Lane 3 untransfected cells
• ss-cDNA single-stranded deoxyribonucleic acid
• Methods and constructs are described that use biological rather than the /// vitro, or artificial, synthesis of ss-cDNA of desired nucleotide base composition Because biological, i e , enzymatic reactions, are used in these methods, they are applicable to any in vivo system
• the expression system of the present invention comprises a vector (as used herein, the term “vector” refers to a plasmid or modified viral or non- viral recombinant biological construct used to deliver and manipulate synthesized and/or naturally occurring nucleic acid sequences) designed to produce any sequence of interest as a ss-cDNA molecule, preferably free of most contiguous vector sequences, within mammalian cells
• the vector system contains all the necessary enzymatic functions and signaling instructions for producing ss-cDNA in the host cell
• the host cell to which the vector of the present invention is delivered produces an RNA transcript (Fig 1 ), driven by an eukaryotic promoter, that is used as a template to direct the synthesis of any desired single-stranded DNA sequence (a "sequence of interest”)
• a first expression system in which the vector comprises two plasmids that are co-transfected into a suitable host cell, which can be yeast or any prokaryotic or eukaryotic cell, to
• the ssDNA produced in vivo using the expression systems described herein may be an inhibitory nucleic acid
• Inhibitory nucleic acids may be ssDNA synthesized from the mRNA template or the mRNA template itself, which can specifically bind to a complementary nucleic acid sequence By binding to the appropriate target nucleic acid sequence, an RNA--RNA, a DNA— DNA, or RNA-DNA duplex or triplex is formed More commonly, these nucleic acids are often termed “antisense” because they are usually complementary to the sense or coding strand of the gene, but the "sense" sequence is also utilized in the cell for therapeutic purposes
• an inhibitory nucleic acid By binding to a target nucleic acid, an inhibitory nucleic acid alters the function of the target nucleic acid This alteration (usually an inhibitory effect) results from, for example, blocking DNA transcription, processing or poly(A) addition to mRNN DNA replication, translation, or promoting inhibitory mechanisms of the cells (such as promoting RNA degradation)
• Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering gene expression
• These different types of inhibitory nucleic acid technologies are described in Helene, C. and J Toulme, 1049 Biochim Biophys Acta 99-125 (1990), hereinafter referred to as "Helene and Toulme,” and which is incorporated herein in its entirety by this specific reference thereto
• inhibitory nucleic acid therapy approaches can be classified into (1) those that target DNA sequences, (2) those that target RNA sequences (including pre- mRNA and mRNA), (3) those that target proteins (sense strand approaches), and (4) those that cause cleavage or chemical modification of the target nucleic acids such as the ssDNA enzymes, including the so-called "10-23 enzyme" as described herein
• the first approach contemplates several categories Nucleic acids are designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors Inhibitory nucleic acids are also used to prevent maturation of pre-mRNA Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation In the second approach, the inhibitory nucleic acids
• inhibitory nucleic acid is the sequence that is complementary to regions of c-myc mRNA, which inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60. which overexpresses the c-myc proto-oncogene (Wickstrom E.
• inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s)
• Inhibitory nucleic acids can also utilize the third approach of designing the "sense" strand of the gene or mRNA to trap or compete for enzymes or binding proteins involved in mRNA translation as described in Helene and Toulme.
• the present invention comprises a set of genetic elements adapted for delivery into a cell to produce ssDNA in vitro or in vivo for altering gene expression, an expression system comprising the set of genetic elements, and one or more stably transfected cell(s) comprising the set of genetic elements
• the set of genetic elements is incorporated into an expression system for delivery into the cell and includes
• RNA dependent DNA polymerase reverse transcriptase
• IR inverted tandem repeat
• PBS primer binding site
• the expression system also preferably includes the functions and signaling instructions for transcription of these components in vivo and the functions and signaling instructions for translation of the reverse transcriptase (RT) gene.
• Additional elements that are optionally included in the set of genetic elements of the present invention may include one or more of an RNAse gene, usually associated with the RT gene, a restriction endonuclease (RE) gene (for a purpose described below), a downstream polyadenylation signal sequence for expression in eukaryotic cells so that the mRNA produced by the sequence of interest includes a poly(A) tail (see Fig 1), and a DNA sequence having enzymatic activity when the linearized ssDNA folds into the appropriate secondary configuration
• the DNA enzymatic sequence is located within a sequence of interest, regardless of whether the sequence of interest is located between the inverted repeat (IR) or between the 3' aspect of the IR and the PBS
• a vector system comprising a two plasmids, and the above-described set of genetic elements that is adapted for delivery to the cell to produce ssDNA /// vivo includes the RNA-dependent DNA polymerase (reverse transcriptase) gene, which additionally contains an RNAse H gene, linked with a histidine-proline linker to a restriction endonuclease gene
• reverse transcriptase RNA-dependent DNA polymerase
• the SOI is located either between the inverted tandem repeats or in a 5' position (with respect to the mRNA transcript) to the PBS, the PBS being located at the most 3' aspect of the mRNA transcript
• the SOI is located (1 ) between the IR, (2) between the IR and the PBS, and/or (3) both between the IR and between the IR and the PBS, and as will be described below, two B plasmids are described herein, one (pssXB-1) with the SOI between the IR (e.g , Notl sites) and the other (pssXB-II) with the SOI between the IR and the PBS (e g , cloned into the PacllBamHl sites)
• plasmid B also includes a combination of transcriptional control elements
• the B plasmid B also includes a combination of transcriptional control elements
• the B plasmid B also includes a combination of transcriptional control elements
• the B plasmid B also includes
• the expression system of the present invention comprises a single plasmid vector, shown schematically in Figs 5 and 6 and designated as plasmids pssXC and pssXD, respectively, in which the above-described set of genetic elements is incorporated
• the resulting mRNA transcript contains the coding region for the RT-RNAse H polyprotein and, at the end of translation at the stop signals, the additional mRNA transcript contains (3' to this translated protein) the elements from the B plasmid with further 3' downstream signaling events for polvadenylation signals, which remain intact from the
• the particular single plasmid expression system described herein does not contain the rest ⁇ ction endonuclease (RE) gene, and therefore does not digest the stem of the stem-loop intermediate formed by the inverted repeats Consequently, the SOI (including the DNA enzyme) is inserted into either the C or D plasmids only in a 3' position to the IR and unwanted vector sequences are removed by premature truncation of the ss-cDNA product as the transcript encounters the relatively stable stem of the stem-loop intermediate and is unable to continue transcribing ss-cDNA from the mRNA transcript More specifically, as will be made apparent in the following description, each SOI was inserted only within the Pacl/BamHl restriction sites of the pssXC and pssXD plasmids
• the plasmids include cloning sites for insertion of the SOI
• Notl sites located between the IR
• Pacl/BamHl 3' to the IR e g , between the IR and the PBS sites are provided in the preferred embodiment of the B plasmid described herein
• the C and D plasmids described herein include onlv the Pacl/BamHl sites for this purpose
• these particular cloning sites were chosen for the particular systems described herein and that other cloning sites may be equally useful for this same purpose
• the A plasmid comprising the two plasmid vector system described herein was not intended to include the SOI, but those skilled in the art will also recognize that, if a two plasmid vector system is to be used, the elements of the set of genetic elements of the present invention, and particularly the SOI. may be inserted
• the nucleic acid sequence that is referred to herein as a cassette provides the template for synthesis of ss-cDNA in target cells It is this element that includes the SOI, IR, and PBS.
• this genetic element is preferably regulated by an appropriate wide spectrum or tissue-specific promoter/enhancer, such as the CMV promoter, or combination of promoters/enhancers, located upstream of the genetic element
• the promoter/enhancer can either be constitutive or inducible promoter Those skilled in the art who have the benefit of this disclosure will recognize that a number of other eukaryotic promoters may be used to advantage to control expression of the SOI including SV-40, RSV (non-cell type specific) or tissue specific glial fibulary acidic protein (GFAP)
• the primer binding site (PBS) for initiation of priming for cDNA synthesis is located between the 3' IR and the polyadenylation signal
• the PBS is a sequence that is complementary to a transfer RNA (tRNA) which is resident within the eukaryotic target cell
• tRNA transfer RNA
• the PBS takes advantage of the proline tRNA
• the PBS utilized in connection with the presently preferred embodiment of the invention that is described herein was taken from the actual 18 nucleotide sequence region of mouse Moloney virus Shinnick, T M., et al., Nucleotide sequence of Moloney murine leukemia virus, 293 Nature 543- 548 (1981)
• the PBS used was taken from the nucleotide sequence of HIV Y.
• any PBS that is matched to a particular RT is utilized for this purpose
• the PBS is exclusively recognized by a primer tRNA that is endogenous to the target cells.
• Each tRNA has the ability to recognize a unique sequence (i.e., codon) on the mRNA transcript coding for an amino acid, and has the ability to covalently link to a specific amino acid (i.e., the tRNA becomes "charged” when bound to a specific amino acid)
• a primer tRNA when bound to the mRNA transcript PBS and not covalently linked with an amino acid (i e , "uncharged", may be used to initiate ssDNA synthesis by the RT.
• the MoMULV RT used in the examples described herein recognizes and uses an uncharged lysine tRNA that in turn recognizes and binds to its unique sequence in the PBS
• each PBS incorporated into the expression system of the present invention must contain the unique sequence recognized by the primer tRNA, and the primer tRNA must be a primer tRNA that is recognized by the particular RT utilized
• RNA-dependent DNA polymerase/RT genes suitable for use in connection with the present invention include those from retroviruses, strains of hepatitis B, hepatitis C, bacterial retron elements, and retrons isolated from various yeast and bacterial species As found in nature, these RNA-dependent DNA polymerases usually have an associated RNase H component enzyme within the same coding transcript However, the present invention does not require the naturally-occurring RNase H gene for a particular RT In other words, those skilled in the art will recognize from this disclosure that various combinations of RT and RNase H genes can be spliced together for use in connection with the present invention to fulfill this function and that modifications and/or hybrid versions of these two enzyme systems are
• the RT/RNase H gene also preferably includes a downstream polyadenylation signal sequence so that the mRNA produced from the RT/RNase H gene includes a 3' poly(A) tail for mRNA stability
• multiple poly(A) tails are available and are routinely used for production of expressed eukaryotic genes
• tissue-specific or wide spectrum promoters/enhancers, or combinations of promoters/enhancers other than those listed above may also be used to advantage to regulate the RT RNAse H gene, the RE gene (if utilized), and the sequence of interest
• the promoters/enhancers may be constitutive or inducible and may include the CMV or
• the RT produced in the cell synthesizes a complementary DNA (cDNA) using as the template the genetic element including the SOI described below
• cDNA complementary DNA
• the RNase H activity of the RT degrades the mRNA template component of the RNA/cDNA hybrid to produce a ss-cDNA in vivo
• the gene encoding the RE (used in the two plasmid expression system and not a required component of that system) may be any of several genes which encode for REs, and preferably those that are controlled by one or more constitutive or inducible wide spectrum and/or tissue-specific promoters/enhancers such as those listed above
• the particular REs tested were Mboll and Fold, but those skilled in the art who have the benefit of this disclosure will recognize that any RE (type I, II, IIS, or III) site may be included in the IR
• These enzymes "clip,” or digest, the stem of the stem-loop intermediate described below to lmea ⁇ ze the SOI as single-stranded DNA
• the RE gene may be regulated by an appropriate constitutive or inducible promoter/enhancer located upstream from the rest ⁇ ction endonuclease gene such as the CMV or RSV promoter for expression in human cells, in plasmid pssXA, the RE gene (Mboll) is linked to the RT-RNAse H polypeptide
• the RE gene also preferably includes a downstream polyadenylation signal sequence so that the mRNA transc ⁇ pt from the RE gene will have a 3' poly(A) tail
• the cassette of the present invention also comprises an inverted tandem repeat (IR) After digestion of the mRNA from the mRNA-cDNA heteroduplex by RNAse H and the release of the ss-cDNA, the IR causes the ss-cDNA to fold back upon itself to form the stem of a stem-loop structure, the stem structure being comp ⁇ sed of double stranded, anti-parallel DNA, in the manner described in U S Patent No 6,054,299 and as shown in Fig 2, after the cassette is transc ⁇ bed in the cell and after the RT RNase H produced by transcription of the genes produces the ss-cDNA sequence of interest from the mRNA transc ⁇ pt in the cell
• One or more RE s ⁇ te(s) which is cut by the RE produced from the RE gene may be designed into the double stranded portion, I e , the IR, that forms the stem of the stem-loop intermediate
• the ss-cDNA which is produced is
• the cassette which comprises one of the elements of the set of genetic elements of the present invention may also include a DNA sequence with catalytic activity
• the present invention is used to particular advantage when the sequence of interest serves as the template for synthesis of an inhibitory nucleic acid that is an antisense sequence
• the examples set out herein describe production of an antisense SOI as set out in Fig 5B including a sequence having enzymatic activity against mRNA including a c-raf cleaving enzyme designed specifically to bind to the 3' untranslated region of the c-ra mRNA, which is targeted by antisense ISIS 5132 (Monia, B P , et al , 2 Nature Medicine 668-675 ( 1996), hereby incorporated into the present specification in its entirety by this specific reference)
• the two 9 bp target specific binding arms were flanked by the 15 b
• the present invention is not limited just to antisense sequences, that the antisense sequence need not necessarily contain a nucleic acid sequence having catalytic activity, and that the inhibitory nucleic acid sequence could also be any of the other types of inhibitory nucleic acid sequences described above
• the above-described SOI was chosen for demonstration of the present invention because the c-raf kinase in A549 lung carcinoma cells system has been well characterized (Monia, et al , supra (1996)) Raf protein is a serine/threonin protein kinase shown to act as a direct downstream effector of ras protein within the MAP kinase signaling pathway with downstream activiation of MEK1/MEK2 and subsequent activiation of ERK1 and ERK2 (Daum, G , et al , The ins and outs of raf kinases, 19 Trends Biol Sci 474-480 (1994)) A number of solid tumors and
• ISIS 5132 phosphorothioate OD ⁇ ISIS 5132
• ISIS 5132 has been shown to induce apotosis (Lau, Q C , et al., 16 Oncogene 1899-1902 (1998), also incorporated into the present specification in its entirety by this specific reference) and appears to represent a potential effective treatment against such tumors
• This antisense OD ⁇ has recently entered Phase I clinical trials (O'Dwyer, P J , et al .
• SOIs that have been cloned into plasmids for expression using the expression system of the present invention include the partial sequence of the 23 rd codon of h-ras antisense binding sequence with the 10-23 D ⁇ A enzyme sequence (Santoro and Joyce, supra (1997)) inserted between the 5' and 3' complimentary sequences, the partial sequence of pleiotropin antisense binding sequence with the 10-23 D ⁇ A enzyme sequence inserted between the 5' and 3' complimentary sequences, and the partial sequence of tat antisense binding region of the SIV sequence with the 10-23 DNA enzyme sequence inserted between the 5' and 3' complimentary sequences Although each of these sequences included the DNA enzyme sequence, those skilled in the art
• the nucleic acid sequence having enzymatic activity utilized in the method of altering gene expression described herein is the 10-23 DNA enzyme (Santoro and Joyce, supra (1997))
• the enzymatic sequence is inserted into the cassette in either or both of the two locations, e g , (a) between the IR and inside the SOI (at the Notl site) or (b) inside the second SOI that is located 3' to the IR and 5' to the PBS (at the PacllBamHl sites) Either way, the resulting aptamer is specific for the target of the SOI and is therefore used to target other D ⁇ A sequences, mR ⁇ A sequences, and any other suitable substrate, to inhibit or change D ⁇ A or mR ⁇ A splicing mechanisms, or even to directly alter the cellular genome in a specific manner
• sequences with DNA ligase activity such as copper-dependent DNAse (Breaker, R R , 97 Chem Rev 371 -390 (1997)) and zinc-dependent E47 ligase (Cuenoud. B and J W Szostak, 375 Nature 61 1-613 (1995)), sequences with DNA kinase acitivity such as calcium-dependent DNA kinase (Li, Y and R R Breaker, 96 Proc Natl Acad. Sci USA 2746-2751 (1999)), and sequences with RNA kinase acitivity such as calcium-dependent DNA kinase (Li, Y , supra ( 1999))
• the vector When the elements comprising the set of genetic elements of the present invention are incorporated into a vector for expression in a target cell, it is preferred that the vector contain other specialized genetic elements to facilitate the identification of cells that carry the vector and cassette and/or to increase the level of expression of the set of genetic elements comprising the cassette
• the specialized genetic elements include selectable marker genes so that the vector can be transformed and amplified in a prokaryotic system
• the most commonly used selectable markers are genes that confer to the bacteria (e g , E.
• the vector contain specialized genetic elements for subsequent transfection, identification and expression in eukaryotic systems
• multiple selection strategies e g , Chinese Hamster O a ⁇ an CHO
• Selectable markers used in eukaryotic systems include, but are not limited to, resistance markers for Zeocin, resistance to G418, resistance to aminoglycoside antibiotics, or phenotypic selection markers such ⁇ -gal or green fluorescence protein Incorporation of these components into the plasmids comprising the expression system of the present invention allows two convenient methods for removing predetermined vector sequences after the production of ssDNA
• the cassette is reverse transcribed from the PBS and the
• the cassette is reverse transcribed from the PBS and an SOI included in the cassette 3' to the IR is likewise transcribed, but reverse transcription is terminated at the stem of the stem-loop structure formed by pairing of the nucleotides of the IR Either way, the resulting ssDNA is produced without (and/or with minimal) flanking sequences
• the cassette is designed with an IR that forms a stem that is more stable than the stem needs to be if the ssDNA is produced by digestion of the stem in accordance with the first aspect of the present invention (for instance, by designing the IR so as not to include an RE site)
• reverse transcription proceeds right on through the second SOI (if it is even designed into the cassette) to the SOI located between the IR This "premature termination" of the reverse transcriptase cDNA transcript at the 3'
• the mechanism involves inhibition of the function of the transc ⁇ ption factor family AP-1 Off ⁇ nga, et al , 62 Cell 527-538 (1990)
• Any desired nucleotide sequence can be inserted into the genetic element that encodes the "loop ' portion of the stem-loop intermediate to carry out a desired inhibitory function, e g , antisense binding, down regulation of a gene, and so on as herein described
• the present invention is used to construct complex secondary ssDNA structures that confer biologic reactions on the cDNA transc ⁇ pt based on conformational secondary structure folding
• Such secondary structure can be engineered to serve any of several functions
• the sequence of interest may include (but is not limited to) a sequence that is incorporated into the loop portion of the single-stranded cDNA transcript to form so-called clover leaf or "crucible" -like structures such as those found in the long terminal repeats of adeno-associated virus or in retrotransposons Under correct circumstances, such structure is integrated in site-specific manner into the host genome
• the cassette of the present invention is adaptable for incorporation into multiple commercially available delivery vectors for mammalian and human therapeutic purposes, multiple delivery routes are feasible depending upon the vector chosen for a particular target cell
• viral vectors are frequently used for transforming the patient's cells and introducing DNA into the genome
• viral vectors carrving new genetic information are used to infect target cells removed from the body and the infected cells are then re-implanted (I e , ex vivo)
• Direct in vivo gene transfer into postnatal animals has been reported for formulations of DNA encapsulated in hposomes and DNA entrapped in proteohposomes containing viral envelope receptor proteins Nicolau, et al , 80 Proc Natl Acad Sci USA 1068-1072 (1983), Kaneda, et al , 243 Science 375-378 (1989), Manmno, et al , 6 Biotechniques 682-690 (1988)
• Positive results have also been described with calcium phosphate co- prec
• the cassette when inserted into the expression system of choice is also advantageously administered through topical, transmucosal, rectal, oral, or inhalation-type methods of delivery.
• the cassette of the present invention is advantageously employed to deliver anti-sense, triplex, or any other inhibitory nucleic acid or single-stranded nucleotide sequence of interest, using known digestion and ligation techniques to splice the particular sequence of interest into the cassette (between inverted tandem repeats or between PBS and inverted tandem repeats).
• digestion and ligation techniques to splice the particular sequence of interest into the cassette (between inverted tandem repeats or between PBS and inverted tandem repeats).
• a likely modification is to change the promoter so as to confer advantageous expression characteristics on the cassette in the system in which it is desired to express the sequence of interest.
• promoters and other signals are so dependent on the particular target cell for which the sequence of interest has been selected, that it is impossible to list all the potential enhancers, inducible and constitutive promoter systems, and/or poly(A) tailing systems which may be preferred for a particular target cell and sequence of interest.
• the present invention takes the form of a kit comprised of a plasmid having the above-described RNA-dependent DNA polymerase and RE genes cloned therein as well as a multiple cloning site (MCS) into which the user of the kit inserts a particular SOI.
• MCS multiple cloning site
• the cloning site into which the SOI is inserted is located between the above-described IR.
• the resulting plasmid is then purified from the cell culture in which it is maintained, lyophilized or otherwise preserved for packaging and shipping to the user.
• the kit preferably also includes the RE(s) for the MCS into which the SOI is to be cloned, the ligases and other enzymes, along with suitable buffers, for ligating the SOI into the plasmid, and a map of the plasmid.
• the SOI(s) is/are delivered to a host cell either by co-transfection of the cells with two plasmids, designated A and B, each plasmid being designed and constructed to include the components listed above, or by a single C or D plasmid
• the B plasmid encodes the cassette including the SOI, either nested within flanking sequences that include the ER or between the IR and the PBS that provides the post-transcriptional processing signals that mediate the conversion of the mRNA into ssDNA.
• the B plasmid includes cloning sites (Notl sites were utilized in the B plasmid described herein) between which any D ⁇ A SOI is placed (as noted above, in the examples described herein, the SOI is an antisense sequence to c-raf kinase including the 10-23 enzyme sequence, but as described above, other sequences that have been produced m vivo using the expression system described herein include a "stuffer," or test, sequence, telomeric repeats, h-ras, a region encoding the angiogenic growth factor pleiotrophin, and the region encoding tat (from SIV))
• Flanking the cloning sites are signals directing the processing of the primary mR ⁇ A transcript, produced from a promoter (a CMV promoter was utilized in the B plasmid described herein), into the desired single-stranded inhibitory nucleic acid After cloning the desired SOI into the B plasmi
• RT reverse transcription of the plasmid RNA transcript by RT, which in the embodiments described herein is an RT expressed by the A, C, or D plasmid (in the embodiment described herein, the RT is MoMuLV RT), proceeding from the primer binding site lying 3' to the SOI (the SOI optionally including the sequence with enzymatic activity), IR, and PBS as shown in Fig
• RNAse H digestion of the resulting heteroduplex either by RNAse H activity of the RT polyprotein or by endogenous RNAse H activity, to release the single-stranded DNA precursor from its RNA complement, and
• the plasmid pcDNA3 lZeo+ was purchased from Invitrogen Corp (Carlsbad, CA) and plasmid pBK-RSV from Statagene (La Jolla, CA) Oligodeoxynucleotides (ODN) were synthesized by Midland Certified Reagent Co (Midland, TX) Polymerase chain reactions (PCR) were carried out using Taq DNA polymerase purchased from Boehringer Mannheim Corp (Indianapolis, IN) in a Robo-gradient thermal cycler (Stratagene (La Jolla, CA)) Restriction endonucleases and T4 DNA ligase were obtained from Boehringer Mannheim Corp (Indianapolis, IN) The ODNs used are listed in the attached Sequence Listing
• plasmids The construction of four expression plasmids is described The first plasmid, pssXB (Fig 3), was derived from pcDNA3 lZeo(+) (Invitrogen Corp ) and contains the genetic element which encodes the ss-cDNA sequence of interest used herein pcDNA3.1Zeo(+) was digested with restriction endonucleases Hindlll and Notl at positions 91 1 and 978, respectively. The double- stranded linker region having compatible HincTAl and Notl ends which is formed by annealing the synthetic, single stranded oligodeoxynucleotides OD ⁇ -5'- ⁇ /M(link)2- H/N and ODN-3'-N M(link)2-H/N was ligated under standard conditions into the Hindill/Notl double-digested pcDNA3 lZeo(+) transformed into Surell cells (Stratagene, Inc ) The ODNs were allowed to hybrid
• the second plasmid is also a component of the two plasmid vector system described herein, pssXA (Fig 3)
• This "A" plasmid contains the Mo-MuLV-RT (Shinnick, T M , et al , 293 Nature 543-548 (1981)) and restriction endonuclease genes and was derived from pBK-RSV (Stratagene), also using XL-1 Blue MRF' as the host cell
• pBK-RSV (Stratagene)
• XL-1 Blue MRF' A mouse cell line expressing Moloney murine leukemia virus was obtained from the American Type Culture Collection (#CRL-1858)
• Viral RNA was isolated from cells in accordance with the method described in Chomczymski, P and N Sacchi (162 Anal Biochem 156-159 (1987)) using Trizol reagent (GibcoBRL) and reverse transcribed using primer -RT-Hmdlll (5'- CTTGT
• the peptide encoded by this construct includes part of the mtegrase gene, which follows the RT in the MoMuLV polyprotein (Petropoulos, supra (1997))
• the bacterium Moraxella bovis which encodes the restriction endonuclease Mboll (Bocklage, H , et al , 19 Nucleic Acids Res 1007-1013 (1991)), was obtained from the American Type Culture Collection (ATCC# 10900) Genomic DNA was isolated from M bovis using the Stratagene DNA extraction kit following the manufacturer's instructions and used as the template DNA in the PCR Using two p ⁇ mers, 5'-Mboll-Hmdlll (5'-CAATTAAGGAAAGCTTTGAAAAATTATGTC-3') and -Mboll-Xmal (5'-TAATGGCCCGGGCATAGTCGGGTAGGG-3'), the Mboll gene was PCR amplified from genomic DNA for 30 cycles 94°C, 30 sec , 58°C, 1 min , 72°C, 1 min These primers were designed to include a Hindlll and an Xmal site, respectively The 1 2 kb product, copying the M bovis genome between positions 888 and 2206
• plasmid pssDNA-Express-A was digested with Sad Xmal to remove the Mboll gene
• a linker region comprised of oligonucleotides 5'-(link)2-Hind/Xba (5 1 - CCGGATCTAGACCGCAAG-CTTCACCGC-3') and 3'-(link)2-Hind/Xba (5'- GGTGAAGCTTGCGGT
• sequences of interest were cloned into the multiple cloning site of pssXC by using the BamHl and Pad sites in the multiple cloning site (Fig 5B)
• Four different sequences of interest were synthesized for these constructs as described above, and similar procedures were utilized for inserting each of the four sequences of interest
• Each construct was prepared by allowing the paired oligonucleotides to anneal at 70°C for 15 minutes and cooling to room temperature, followed by ligation into the plasmid under standard conditions After transformation into Surell cells, appropriate colonies were selected with verification by sequencing for the individual inserts
• a second plasmid was constructed for use in a single-plasmid expression system, pssXD, by combining the two plasmids, pssXA and pssXB in the following manner pssXA, which contains the Mo-MuLV reverse transcriptase (RT), was digested with Xmal and Bgtll and the resulting
• reverse transcriptase activity was assayed using the RT-PCR assay developed by Silver, J . et al (An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions, 21 Nucleic Acids Res 3593- 3594 (1993)), with modifications as set out below pssXA transfected cells were lysed with lysis buffer (1% TritonTM, 1 mM MgCl 2 , 100 mM NaCl, 10 mM TRIS-HCl, pH 8 0 and 2 nM DTT) After centrifugation at 18,000g for 30 min , the supernatant was collected and frozen at -80°C until use Brome mosaic virus (BMV) RNA, used as a template, was reverse transcribed by incubation with the lysate, which would contain RT activity, for 1C or 30 min at 37°C Using primers 5'- CGTGGTTGACACGCAGACCTCTTAC-3' and 5
• This RT-PCR assay relies upon RT activity in the cell lysates of transfected cells to produce a cDNA transcript of the BMV RNA substrate
• the replication cycle of this virus does not involve a DNA intermediate, eliminating the possibility that an amplification product could be produced without prior reverse transcription RT activity was determined in the lysates of A549 cells transfected with the pssXA plasmid (lanes 3 and 4) and the E10 clone, which showed relatively high expression (lanes 5 and 6) RT activity was also determined from A549 cells transiently transfected with control pBK-RSV plasmid (lanes 1 and 2)
• lysates were prepared 48 hours after transfection Results show that cell lysates from both transient and stable transfected (E10) cells support the production of a band of expected size, 150 bp (lanes 3-6), whereas control lysates showed none (lanes 1 and 2)
• c-raf DNA enzymes were further confirmed by dot-blot detection of ssDNA, using the North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Pierce) following the manufacturer's instructions Two ⁇ g of total RNA, isolated from cells transfected with either pssXA pssXB-I or pssXA pssXB-II, or pssXA or untransfected cells, was used The sequence of c-raf specific, biotin-labeled probe is 5'-
• a biotin-labeld c-raf specific oligo probe can only detect signal in the RNA preparations isolated from ElO cells transfected with pssXB-I or pssXB-II but not untransfected ElO cells or A549 cells
• Assays for ssDNA were performed by PCR and by dot-blot analyses 24-48 hours after transfection as described above Reverse transcriptase activity was assayed using the RT-PCR assay developed by Silver, et al (supra (1993)) also as described above Individual colony isolates of stably substituted HeLa cell lines (A12 and B12) were additionally assayed for RT activity
• the ss-cDNA was isolated from cells transfected 48-72-hr earlier
• the ss-cDNA which co-localizes with RNA, was earned out using T ⁇ zol reagent (Gibco Life Technologies, Gaithersburg, MD)
• Assays for specific ss- cDNA species were carried out bv both PCR based assays for internal fragment and by denatured single stranded gel electrophoresis with subsequent nylon blotting and probing with an internal biotin-labeled probe This experiment showed that human tissue culture cells (HeLa cell line), transfected with
• RNA samples isolated from A549 cells either transfected with control pssXD-I or pssXD-II containing the c-raf DNA enzyme sequence
• Two ⁇ g of total RNA were pretreated with RNase A to rule out any possible non-specific hybridization to RNA, and in the presence and absence of SI nuclease for 30 min at 37°C Subsequently, samples were loaded onto a Hybond-N+ membrane (Amersham Pharmacia Biotec, Piscataway, NJ), and fixed by UV exposure for 3 min Hybridization and signal detection were performed using the North2South Chemiluminescent Nucleic Acid Hybri
• c-raf mRNA was quantitated by RT-PCR as described by Li, et al (1 Gene Therapy 321-328 (2000)) with some modification Briefly, one ⁇ g of total RNA was reverse transcribed using the Reverse Transcription System (Promega Corp , Madison, WI) A fraction of the resulting cDNA was used as a template for PCR amplification Forty cycles of PCR were conducted (95°C, 30 sec, 50 °C, 30 sec, and 72 °C, 60 sec) using specific primers The specific primer sequences used were as follows 1) c-raf primers: 5'- TCAGAGAAGCTCTGCTAAG-3' and 5'-CAATGCACTGGACACCTTA-3', 2) actin primers- 5'-ACCTTCTACAATGAGCTGCG-3' and 5'-
• RNA isolated from cells transfected with either control pssXD-I or pssXD-II containing c-raf DNA enzyme sequence, was reverse transcripted and PCR amplified using a pair of c-raf specific primers PCR amplification of actin mRNA was used as a control to normalize loading quantity among different samples. As shown in Fig. 12, a significant reduction (approximately 70-80%) of c-raf mRNA was detected in the cells transfected with pssXD-II (Lane 2) compared to that of control (Lane 1 )
• the levels of c-raf protein in A549 cells transfected with either pssXD-I or pssXD-II were assessed by Western Blot analysis 30 ⁇ g of cell extracts were subjected to electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) Proteins were electrotransferred using a Mini Trans-Blot Electrophoretic Transfer Cell according to the manufacture's instructions (BioRad Laboratories, Hercules, CA) to a Hybond ECL membrane (Amersham Pharmacia Biotec, Piscataway, NJ) The membrane was subsequently blocked in a buffer containing 25 mM Tris-HCl, pH 7 5.
• Sequences that act as antisense oligonucleotides to one or more RNA molecules encoding one of the several dopamine receptors for therapy of Parkinson s disease The antisense oligonucleotides bind specifically to expression-controlling sequences of such RNA molecules, thereby selectively controlling expression of one or more dopamine receptor subtypes, and alleviating the pathological conditions related to their expression,
• Oligonucleotides having a sequence of nucleotide bases specifically hyb ⁇ dizable with a selected sequence of a cytomegalovirus DNA or RNA, specifically, sequences targeting cytomegalovirus DNA or RNA coding for the EE1, EE2, or DNA polymerase proteins It is preferred that such oligonucleotides have between about 5 and about 50 nucleic acid base units as described in U S Patent No 5,442,049 Oligonucleotides specifically hybridizable with RNA or DNA deriving from a gene corresponding to one of the open reading frames UL5, UL8, UL9, UL20, UL27, UL29, UL30, UL42, UL52 and IE175 of herpes simplex virus type 1 comprising nucleotide units sufficient in identity and number to effect such specific hybridization.
• the oligonucleotides be specifically hybridizable with a translation initiation site, coding region or 5' untranslated region
• the oligonucleotides are designed to be specifically hybridizable with DNA, or preferably, RNA from one of the species herpes simplex virus type 1 (HSV-1), herpes simplex virus type (HSV-2), cytomegalovirus, human herpes virus 6, Epstein Barr virus (EBV) or varicella zoster virus (VZV)
• HSV-1 herpes simplex virus type 1
• HSV-2 herpes simplex virus type
• cytomegalovirus cytomegalovirus
• ESV Epstein Barr virus
• VZV varicella zoster virus
• herpes simplex virus type 1 find counterparts in the other viruses named
• herpes simplex virus type 2 cytomegalovirus, human herpes virus type 6, Epstein Barr virus and varicella zoster virus
• the present invention is directed to antisense oligonucleotide therapy in which the oligonucleotides are directed to any of the foregoing viruses, or indeed to any similar viruses which may become known hereafter, which have one or more of such analogous open reading frames
• all such viruses are denominated as herpes viruses.
• Antisense oligonucleotides against ICAM-1 gene expression in interleukin-1 beta-stimulated cells for use as anti-inflammatory agents with activity towards a variety of inflammatory diseases or diseases with an inflammatory component such as asthma, rheumatoid arth ⁇ tis, allograft rejections, inflammatory bowel disease, various dermatological conditions, and psoriasis
• inhibitors of ICAM-1, VCAM-1, and ELAM-1 may be effective in the treatment of colds due to rhinovirus infection, AIDS, Kaposi's sarcoma and some cancers and their metastasis as desc ⁇ bed in U S Patent No
• Protein-binding oligonucleotides that specifically bind target molecules such as proteins, and particularly thrombin, in the host cell as described in U S Patent No 5,840,867
• target molecules such as proteins, and particularly thrombin
• the cassette described herein is desc ⁇ bed as comprising three genetic elements, a sequence of interest, a primer binding sequence, and a tandem inverted repeat, and when transfected into a target cell with a reverse transc ⁇ ptase gene under control of a suitable promoter, produces the inhibitory nucleic acid sequence desc ⁇ bed herein
• the mouse Moloney leukemia virus reverse transcriptase gene desc ⁇ bed for use as the reverse transcriptase gene of the cassette can be replaced with other reverse transc ⁇ ptase genes (the reverse transc ⁇ ptase gene from human immunodeficiency virus was one such gene which was noted above) and that promoters other than the CMV promoter described herein may be used to advantage.
• the stem-loop intermediate that is formed may or may not include a restriction endonuclease site and its susceptibility to denaturation is manipulated to advantage depending upon the particular sequence of interest that is intended to be produced from that intermediate. All such changes, and others that will be made clear to those skilled in the art by this description modifications which do not depart from the spirit of the present invention, are intended to fall within the scope of the following claims.

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